Effects of Seed Weight and Substrate on Germination and Growth of Non-toxic Jatropha curcas L....

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*Corresponding author: Email: [email protected];

Annual Research & Review in Biology4(24): 4232-4245, 2014

SCIENCEDOMAIN internationalwww.sciencedomain.org

Effects of Seed Weight and Substrate onGermination and Growth of Non-toxic

Jatropha curcas L. Seedings

Ofelia Andrea Valdés Rodríguez1, Arturo Pérez Vázquez2*

and Armando J. Martínez3

1Research Line 3, Colegio de Postgraduados, Mexico.2Research Line 3, Colegio de Postgraduados, Mexico.

3Instituto de Neuroetología,Universidad Veracruzana, Mexico.

Authors’ contributions

This work was carried out in collaboration between all authors. Author OAVR designed thestudy, performed the statistical analysis, wrote the protocol and wrote the first draft of themanuscript. Author AJM managed the analyses of the study and the Figures. Author APVreview and corrected the manuscript; provided de genetic material, the lab equipment andeconomic resources for the research. All authors read and approved the final manuscript.

Received 29th May 2014Accepted 8th July 2014

Published 3rd August 2014

ABSTRACT

Aims: We characterized the responses of non-toxic seedlings of Jatropha curcas L. tovariation in seed weight and substrate during their germination and growth.Study Design: Two analytical experiments using a randomized controlled trial approachwere applied to consider the effects of seed weight and substrate.Place and Duration of Study: Non-toxic seeds from the east coast of Mexico wereselected during two consecutive years and their seedlings were studied during one monthafter emergence.Methodology: The seeds were grouped into different weight categories (400-499 - 800-899 mg). The substrates evaluated were sandy, sugarcane compost and a mix of sandyand sugarcane compost (sandy-compost, 2:1). While sandy substrate was poor in organicmatter (OM), sugarcane compost was rich in OM. Germination, survival rate and meangermination time (MGT) were evaluated by seed weight category and substrate. Wemeasured stem length, root collar diameter, number of leaves and leaf area every week,

Original Research Article

Annual Research & Review in Biology, 4(24): 4232-4245, 2014

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and at the end of the experiments total dry biomass was recorded.Results: The heaviest seeds had higher germination rates (86.3%±7.1%) compared tothe lightest seeds (69.9%±12.2%) in all substrates. Survival rate was 10% lower for thelightest seeds in sandy-compost and for the heaviest seeds in compost. MGT was notsignificantly different between seed weight categories or substrates (P=.32). Heavierseeds produced bigger seedlings in terms of stem length, root collar diameter, number ofleaves, root length, and total dry mass in sandy-compost (P<.05). However, whensubstrates were compared, the substrate effect was larger than the effects from seedweight, with the largest seedlings found in compost (P<.05), regardless of seed weight.Conclusion: Seed weight improved germination rates in sandy and sandy-compostsubstrates. Compost increased seedling growth regardless of seed weight.

Keywords: Jatropha curcas; seed weight; substrate; germination; seedling growth.

1. INTRODUCTION

The promising biofuel source Jatropha curcas is still under domestication [1,2]. Therefore,agronomic practices are being explored. For the Mexican non-toxic Jatropha curcas,scientific information is scarce. Seeds from this plant contain very low or non-detectablephorbol esters [3], the main toxic compounds widely distributed in species of theEuphorbiaceae [4]. Scientific research on J. curcas seed performance during propagation islimited [5–9]. Some field experiences in commercial plantations indicate that bigger andheavier seeds show higher viability and vigour compared with smaller and lighter seeds[10,11]. Another significant factor for seed germination and satisfactory seedlingestablishment in the field is the type of substrate. For J. curcas, studies relating germinationand survival in various substrates suggest that sand can provide high germination rates[5,8,12]. However, research with other substrates and their relationships with seed weightare not available, and although there are suggestions that the plants grow well in sandy-loamy (aerated) soils; some researchers have shown that J. curcas could have very limitedgrowth in poor sandy soils [13,14]. Therefore, there is a need to know the effects of seedweight and substrate on germination and growth of seedlings. The objectives of thisresearch were to determine the effects of seed weight distribution and type of substrate on1) Germination and survival rate, and 2) Seedling growth and biomass production of non-toxic Jatropha curcas seedlings.

2. MATERIALS AND METHODS

2.1 Plant Material and Seed Characterization

Native seeds from two sources along the Gulf coast of Mexico were collected. Oneprovenance from the municipality of Medellín (18º59’52” N, 96º15’31” W; 17 masl) wascollected during July 2009, and one provenance from the municipality of Alvarado (18º46’26”N, 95º45’35” W; 4 masl) was collected during August 2010. Mother plants were part of livingfences without irrigation or fertilization. Both sources were located in the same geographicalregion, “Sotavento”. The climate in the region is warm sub-humid (Aw2, Köppen classification[15]) with minimum and maximum temperatures of 20 and 29ºC, respectively, and an annualrainfall of 1500 mm (concentrated during one rainy season from June to October) and onedry season from November to May); and the region contains regosolic soils [16]. For eachcollection, all ripe yellow fruits were harvested and the seeds obtained manually. The seeds


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were dried in a shaded and aerated place until they were completely dry. Prior to theexperiment, seeds were kept inside glass bottles in a cool and dry location (18ºC).

Phorbol ester contents in the seeds were determined following [17] and did not detect anyphorbol 12-myristate 13-acetate (PMA) from the Alvarado provenance, and detected only0.19mg·g-1 from the Medellin provenance. Both provenances were considered non-toxicbecause local people consume cooked seeds. Seed oil content was estimated as 61% forMedellin and 54% for Alvarado.

2.2 Seed Weight Frequency Distribution and Seed Selection

All mature fruits from each selected shrub in each provenance were collected. Thedistribution of seed sizes was obtained by measuring seed length and width using a digitalAutotec caliper (0.01 mm accuracy, range 0-150 mm) and weight on an Ohaus PioneerPA64C analytical balance (0.0001 g precision, range 0-65 g).

2.3. Experimental Design

Two experiments were conducted: experiment 1, during May 2010, which evaluated thecollection from Medellin, and experiment 2, during October 2010, which evaluated thecollection from Alvarado. Based in their weight distributions and availability per collection(Fig. 1), the seed weight categories were conformed to equal seed quantities in eachcategory. Experiment 1 consisted of 51 seeds per category and experiment 2 consisted of32 seeds per category. Experiment 1 evaluated seed weight categories in one substrate, amix 2:1 of sandy soil and compost made of sugarcane bagasse (obtained from a local sugarmill) and labeled as sandy-compost. Experiment 2 evaluated seed weight in two substrates:A mix of 1:1 sandy and sandy-loamy soil, labeled as Sandy; and pure sugarcane bagassecompost, labeled as compost. One seed was sown per black polyethylene bag (30 cm heightand 15 cm diameter). Seeds were placed in vertical position with caruncle down and at adepth of 2 cm. The treatments were identified with permanent ink on the bag and randomlydistributed under a mesh shade. Seeds and emerging seedlings were watered daily until theend of the experiments.

2.4 Experimental Conditions

The experimental site was located in the municipality of Manlio Fabio Altamirano, Veracruz,which also belongs to the Sotavento region (19º16'00" N, 96º16'32" W; 16 masl), with thesame climate (Aw2). The experimental units were placed under a mesh-shade of 25%. Plantmaterial and site conditions registered during experiments are summarized in Table 1.

Table 1. Environmental conditions during each experiment

Experiment Minimum averagetemperature ± S.D.* (ºC)

Maximum averagetemperature ± S.D. (ºC)

Relative humidity ±S.D. (%)

Experiment 1 23.7±1.4 34.4±1.9 74.8±5.9Experiment 2 19.8±1.1 30.2±1.5 71.5±7.1

*S.D. = Standard deviation


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2.5 Substrate Analysis

The substrates were analyzed for pH (pH meter), nitrogen (semi-micro-Kjeldahl method),potassium (PFP7 Flame Photometer) and phosphorus (Olsen method).

2.6 Variables Measured

2.6.1 Germination

Germination was assessed from the second day until day 20 when no further germinationwas observed. Survival was assessed as the number of seedlings surviving after a period of20 days following their germination.

2.6.2 Mean germination time

Mean germination time (MGT) was calculated according to [18]:

MGT=∑i=1→k(niti)/∑ i=1→k(ni) (1)

Where

ni = The number of seeds germinated only during day i; ti = the number of days for ni;k = Time of the last germination event.

2.6.3 Measurements

The following variables were measured every week: shoot length (using a tape measurehaving 1 mm precision), measured as the distance between the root collar and the apicalmeristem and root collar diameter (using a digital caliper having 0.01 mm precision). At theend of the experiments, the number of leaves and the area of the first true leaf (using a tapemeasurement with 1 mm precision) were calculated as the product of 0.84*(leaf length*leafwidth)0.99[19]. Later, the plants were uprooted, the roots washed, and the whole plant wasoven-dried at 70ºC for 72h to determine plant dry mass (using an analytical balance 0.0001g precision). For experiment 2, additional to these variables, the fresh tap-root and four mainlateral roots were measured for diameter at the base and at the tip of each root (digitalcaliper, 0.01mm precision) and length (tape measure, 1 mm precision). With this data thecoarse root volume was estimated.

2.7 Statistical Analysis

Survival analyses were applied using the Kaplan-Meier Log-Rank test to evaluate theproportion of germination and survival seed rate in both experiments. The MGT, stem length,root collar diameter, area of the first true leaf and biomass were compared using analyses ofvariance (ANOVA). Normality and equal variance tests were conducted and normallydistributed data were analyzed with parametric tests, while non-normally distributed datawere analyzed by applying the non-parametric Kruskal-Wallis test on ranks. Experiment 2,with two factors (germination and vigour by seed weight category and substrate), wasanalyzed using a two-way ANOVA. Post hoc multiple comparisons were performed using theHolm-Sidak method, at a level of significance of α=.05. Growth variables were compared


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weekly and at the end of the two experiments. Sigma Plot version 10.0 was used for allanalyses.

3. RESULTS

3.1. Seed weight Frequency Distribution

Fig. 1 shows the frequency distribution of weight and size from the two provenances. Seedsfrom Medellin had a median of 623 mg and seeds from Alvarado a median of 700 mg. Inboth provenances, more than 68% of the weights were located in the range of 600 to 799mg. Seed weight distribution from Medellin provided categories from 400 to 799 mg, whileAlvarado provided categories from 500 to 899 mg (Table 2).

Table 2. Weight categories and their average weight, length and width (±S.D.) obtainedfrom the provenances of Medellin and Alvarado

Experiment and provenance Seed weightcategories (mg)

Weight(mg)

Length(mm)

Width(mm)

Experiment 1(Medellin)

400-499500-599600-699700-799

453.9±34.0545.3±21.6620.6±11.4733.8±24.8

16.3±0.916.7±0.417.8±0.718.6±0.6

8.4±0.58.4±0.29.0±0.39.2±0.3

Experiment 2(Alvarado)

500-599600-699700-799800-899

561.8±32.3648.1±26.9744.5±27.9840.8±31.7

16.3±1.216.8±1.317.6±1.217.8±1.5

9.2±0.69.4±0.79.7±0.510.0±1.0

3.2 Substrate Analysis

Results of substrate analysis are shown in Table 3. All the substrates were located in thealkaline range, being Compost the one with the highest contents of nitrogen, phosphorusand potassium (three times more nitrogen than sandy-compost, and more than ten timesnitrogen than sandy).

Table 3. Physical and chemical properties of the substrates

Substrate pH Nitrogen (%) Phosphorus (ppm) Potassium (mg/L)Sandy-compost 7.35 0.35 20.32 81.34Sandy 7.03 0.09 17.17 9.13Compost 7.01 1.05 43.59 240.70

3.3 Germination and Survival by seed Weight

The proportion of the germination curves showed differences among seed mass categories(K-M Log-Rank = 185.35, P<.001), with the lightest seeds having the lowest germination rate(Fig. 2A). Survival rate was also different between categories, (K-M Log-Rank = 111.02,P<.001), with the lightest seeds having the lowest survival rate. MGT was similarindependent of seed weight (H=3.9, P=.27) (Table 4).


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Fig. 1. Frequency distribution of seed weight, length and width from the provenances of Medellin (A, B, C) and Alvarado(D, E, F)


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3.4 Germination and Survival by Substrate and Seed Weight

Germination curves differed among substrate and weight categories (K-M Log-Rank =175.53, P< .001). The heaviest seeds in sandy soil had the highest germination rate, whilethe lightest seeds in compost had the lowest germination rate (Fig. 3A). Survival rate differedamong substrates (K-M Log-Rank =17.97; P<.001). The heaviest seeds in compost hadlower survival rate compared with categories 600-799 mg (Table 4). The MGT was similarindependent of seed weight and there was no statistically significant interaction betweensubstrate and weight (F=1.089; P =.36) (Table 4).

3.5 Growth Rates by Seed Weight

For experiment 1, the stem growth rate was significantly different among categories(Fig. 2B), except during the second week (H=7.23, P=.06) and, at the end of the third weekseedlings from seeds heavier than 600 mg were taller than those of 400-599 mg (P<.001;Fig. 2b). Growth rate in root collar diameter was different the first week, but similar during thesecond and third weeks (P=.07). These differences, and the small weekly accumulativedifferences, produced wider root collar diameters in seedlings from seeds heavier than 700mg at the end of the experiment (Fig. 2C). The number of leaves during the third week wasgreater in seedlings from seeds heavier than 500 mg compared with lower weights(H = 10.88; P=.013). Leaf area of the first true leaf was similar in the four categories (H =3.22; P=0.36).

3.6 Growth Rates by Seed Weight and Substrate

Within weight categories, there were no significant differences in stem length, root collardiameter, number of leaves or area of the first true leaf (P=.05; Table 5). Although, duringthe second week the lightest category (500-599 mg) had the lowest growth rate in height.

Seedlings in compost had higher growth rates over five weeks. They also had greater stemgrowth rate in height and diameter since the second week and greater leaf area and numberof leaves since the third week until the end of the experiment compared to seedlings insandy soils (Figs. 3B, C). Root volumes were larger in seedlings from compost thanseedlings from sandy substrate during the fifth week. However, longer roots were recordedfrom sandy soils.

Over five weeks there was no statistically significant interaction between weight andsubstrate for stem height (P≥.13). However, during the fourth and fifth week, there was astatistically significant interaction between weight and substrate for stem growth rate(P=.02), being the highest category in compost (800-899 mg) the one with the highest stemgrow rates.

3.7 Dry Mass and Biomass Distribution by Seed Weight

For Experiment 1, dry root mass did not differ significantly among seedlings from differentseed weight categories (Kruskal-Wallis; P=.06). However, stems were heavier in seedlingsfrom seeds weighing more than 600 mg (Kruskal-Wallis; P<.001), and the total dry weightwas higher in seedlings from seeds having weights over 700 mg (Kruskal-Wallis; P<.001)compared with seedlings having seed weights less than 600 mg. The greatest proportion ofbiomass was allocated to leaves during the third week in all seedlings (Table 6), with higher


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biomass in seedlings with seed weights heavier than 700 mg compared to seedlings fromseeds lighter than 500 mg.

Table 4. Germination and survival rate in response to seed weight category andsubstrate

Experiment Substrate Weightcategory (mg)

Germinationrate (%)

Survivalrate (%)

Mean germinationtime

Experiment 1 Sandy-Compost

400-499 60.00b 80.00b 8.52a

500-599 98.04a 88.33a 7.29a

600-699 96.08a 90.00a 7.56a

700-799 90.20ab 85.00ab 7.35a

Experiment 2 Sandy 500-599 62.50b 90.00ab 5.11a

600-699 56.25b 94.40a 3.72a

700-799 62.50b 90.50ab 5.21a

800-899 90.63a 96.60a 4.62a

Compost 500-599 40.63c 92.00ab 4.00a

600-699 46.88b 100.00a 4.06a

700-799 50.00b 100.00a 4.61a

800-899 78.13a 84.00b 3.65a

Different letters within a column and within an experiment indicate a significant difference between treatments(P<.05)

Table 5. Variables measured (±S.D.) by seed weight category at the end of the twoexperiments

Experiment andtreatment

Seed weight(mg)

Stem length(mm)

Root collardiameter (mm)

Number ofleaves

Area of first trueleaf (cm2)

Experiment 1Sandy-Compost 400-499 105.2±12.9c 6.3±0.5c 3.6±0.5b 55.1±23.1a

500-599 122.7±16.5b 6.6±0.6bc 3.9±0.4a 60.2±17.9a

600-699 127.0±14.3ab 6.8±0.7ab 4.0±0.0a 62.0±10.5a

700-799 135.8±20.7a 7.0±0.6a 4.0±0.0a 64.1±9.5a

Experiment 2Sandy 500-599 149.8±31.2b 9.8±1.0b 5.3±0.5b 105.9±31.2b

600-699 163.7±27.9b 9.8±0.7b 5.6±0.5b 106.1±24.5b

700-799 162.6±23.4b 10.7±0.9b 5.4±0.7b 120.0±32.5b

800-899 152.3±26.2b 11.1±1.4ab 5.6±0.9b 123.3±22.8b

Compost 500-599 208.6±49.6a 11.4±1.4a 7.0±1.3a 151.2±41.4a

600-699 239.4±44.7ª 11.1±1.3a 7.2±1.7ª 155.8±34.9a

700-799 209.5±28.7a 11.7±0.9a 6.9±1.1ª 148.2±32.1a

800-899 236.4±50.1a 11.8±1.4a 7.1±1.1ª 149.6±36.6a

Different letters within a column and within an experiment indicate a significant difference between treatments(P<.05)

3.8 Dry mass and biomass distribution by seed weight category and substrate

For experiment 2, the effects of seed weight on their seedlings were statistically similar forstems (P=.13), leaves (P=.08) and roots (P=.22). There was no statistically significantinteraction between seed weight and substrate for stems (P = .43), leaves (P =.82), or roots(P=.29). However, compost produced the heaviest stems and roots (P<.001). The root toshoot ratio was greater in plants from sandy soil compared to compost (P=.01), and therewas no statistically significant interaction between seed weight and substrate (P =.61).


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Fig. 2. Germination and growth curves from Experiment 1Mean ± S.E.M = Mean values ± Standard error of means of four weight categories


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Fig. 3. Germination and growth curves from experiment 2Mean ± S.E.M = Mean values ± Standard error of means of four weight categories


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Table 6. Average dry mass and root to shoot ratio (±S.D.)

Experiment andtreatment

Seed weight(mg)

Dry mass (mg) Root to shootratioRoots Stem Leaves

Experiment 1Sandy-Compost 400-499 236±64a 325±64b 509±116c 0.29±0.07a

500-599 206±81a 341±81b 509±73c 0.25±0.07a

600-699 194±105a 393±105ab 613±94b 0.19±0.06b

700-799 218±62a 468±157a 739±118a 0.18±0.03b

Experiment 2Sandy 500-599 388±194a 2116±189b 1382±148b 0.11±0.05a

600-699 373±67a 2214±189b 1472±148b 0.10±0.01a

700-799 482±355a 2667±179b 1702±140b 0.12±0.11a

800-899 479±184a 2567±174b 1820±136b 0.11±0.02a

Compost 500-599 438±115a 2953±208a 2215±163a 0.09±0.02ab

600-699 453±119a 2826±184a 2357±144a 0.09±0.04ab

700-799 405±112a 2902±189ab 2345±148a 0.07±0.01b

800-899 498±120a 3201±184a 2511±144a 0.09±0.02ab

Different letters within a column and within an experiment indicate significant differences betweentreatments (P<.05)

4. DISCUSSION

4.1 Germination and Survival Rates by Seed Weight and Substrate

Seed weight influenced germination rates in J. curcas. Heavier seeds yielded highergermination percentages in the three substrates. These results are congruent with fieldexperiences reported for toxic seeds of this species [10,11]. The lower germination in thelightest seeds from each provenance is also an indication that seeds with low weights atcollection may not have enough maturity and energy to achieve germination [20]. Therefore,it can be considered that greater energy content in heavier seeds contributed to improvegermination rates [21]. It should be noted that seeds from Medellin had 7% more oil thanseeds from Alvarado and showed higher germination rates for the same weight categories(500-799 mg), although higher temperatures may also have favored this improvedperformance, since they were close to optimum temperatures reported for Jatropha [5,22].

Weight distribution also played a role in survival rate, since higher survival occurred forweights with the highest frequency of distribution (Table 4). Survival percentages accordingto their distribution of weights are considered a survival strategy for the most abundantseeds [23].

When compared, sandy soil was better than compost for seed germination. Bettergermination performance in sandy soils and soils with low organic matter have already beenreported for toxic and non-toxic provenances [5,8]. Compost has a higher moisture retentionand, by being rich in organic matter, favors the development of bacteria and fungi that attackseeds; while sandy soils, which are low in organic matter and provide better aeration for theseeds, favor germination [24,25].


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4.2 Growth rates by Seed Weight and Substrate

In sandy-compost substrate, the heavier the seed, the larger the size of the plant resulting atthe end of the third week, indicating that seed biomass affected stem growth. Seed weightalso positively influenced root collar diameter in sandy, a poor organic matter substrate, withlow nitrogen content. However, when soil was rich in organic matter, with higher nitrogen,phosphorus and potassium levels, the effects of seed weight were not significant. A richersubstrate like compost significantly increased above and below-ground developmentcompared to poor sandy substrates; however, the root system of J. curcas extended itscapabilities to reach more area in this arid environment, and roots were longer in seedlingswith heavier seed weights. This is an indication that seed weight positively affects the abilityof J. curcas to survive in low-nutrient environments [26,27]. Additionally, larger and heavierseeds of J. curcas have been associated with better field performance and higher yieldpotential [6].

4.3 Biomass by Seed Weight Categories and Substrate

In sandy-compost substrate, biomass of leaves and stems was influenced by seed weight;heavier seeds produced more stems and leaf biomass. Yet, seedlings from smaller seedshad higher root: shoot ratios, compensating for the lower energy content in their cotyledonswith a higher ability to explore for external resources. When seedlings germinated and grewunder conditions such as with the sandy and compost media, substrates played a larger rolethan seed weight regarding biomass production. Thus, seedlings developing under a richsubstrate will have similar biomass independently of their seed weight. The bigger root toshoot ratio in poor substrates, compared with richer substrates has been previouslyobserved in juvenile seedlings of Jatropha [14]. This performance could be considered astrategy to improve foraging outcomes for soil resources, which is an adaptive survivalstrategy of species adapted to nutrient-poor soils [28,29].

5. CONCLUSION

There is a strong interactive effect of seed weight on germination and growth of non-toxicJ. curcas seedlings. Heavier seeds perform better in soils with little organic matter.Substrates rich in organic matter (like compost) favor seedling growth and biomassproduction independently of seed weight. It is recommended to select heavier seeds forplanting in soils rich in organic matter to obtain healthier and larger plants.

ACKNOWLEDGEMENT

Prioritary Line of Research (LPI): 3; Research funded by a CONACyT postdoctoralscholarship assigned to 65556 and the project PFA C-703/2013 (UV) provided to AJM.

COMPETING INTERESTS

Authors have declared that no competing interests exist.


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_________________________________________________________________________© 2014 Rodríguez et al.; This is an Open Access article distributed under the terms of the Creative CommonsAttribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly cited.

Peer-review history:The peer review history for this paper can be accessed here:

http://www.sciencedomain.org/review-history.php?iid=582&id=32&aid=5628

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